Characterization and functional assay of apsup (Lyxy105) from Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV)

Abstract

The baculoviral anti-apoptotic genes, p35 and iap (inhibitor of apoptosis), play important roles in the initiation of viral infection. Recently, a new anti-apoptotic gene (apoptosis suppressor, apsup) was identified in Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). An apsup homolog gene, Lyxy105 (ly-apsup), was also predicted in the Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) genome. In this study, we attempt to perform a gene expression analysis and a functional assay of ly-apsup to demonstrate its anti-apoptotic activity and identify the functional domain of this protein. The transcription of the ly-apsup gene region was detected from 12 h post-infection (hpi) and increased significantly after 24-72 hpi. Comparison of the putative amino acid sequences to those of 18 baculoviral homolog proteins showed high amino acid identity to the LdMNPV sequences. Moreover, five conserved protein domains (named as domains I-V) were found. Therefore, protein functional assays were conducted on full-length proteins and different truncation clones. The overexpression of each clone was confirmed by western blot analysis, and the data revealed that a cleavage of ~ 5 kDa at the N-terminal region of the full-length, domains I-IV (1-241) and I-III (1-178), proteins occurred. The results of the functional analysis showed that full-length Ly-apsup and Ly-apsup with domain I (1-70) could inhibit Drosophila-RPR protein (D-RPR)-induced and actinomycin D (ActD)-induced apoptoses. In addition, the domains I and I-II (1-126) regions showed higher anti-apoptotic activity than the other domains in both D-RPR-induced and ActD-induced cell apoptoses. In conclusion, domain I of Ly-apsup may play an important role in the anti-apoptotic activity of this protein; cleavage of the Ly-apsup N-terminus may lead to decreased anti-apoptotic activity.

Keywords: Apoptosis; Apsup; Drosophila rpr; Functional assay; LyxyMNPV.